Natural-Product-Inspired Microwave-Assisted Synthesis of Novel Spirooxindoles as Antileishmanial Agents: Synthesis, Stereochemical Assignment, Bioevaluation, SAR, and Molecular Docking Studies

Leishmaniasis is a neglected tropical disease, and there is an emerging need for the development of effective drugs to treat it. To identify novel compounds with antileishmanial properties, a novel series of functionalized spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one 23a–f, 24a–f, and 25a–g were prepared from natural-product-inspired pharmaceutically privileged bioactive sub-structures, i.e., isatins 20a–h, various substituted chalcones 21a–f, and 22a–c amino acids, via 1,3-dipolar cycloaddition reactions in MeOH at 80 °C using a microwave-assisted approach. Compared to traditional methods, microwave-assisted synthesis produces higher yields and better quality, and it takes less time. We report here the in vitro antileishmanial activity against Leishmania donovani and SAR studies. The analogues 24a, 24e, 24f, and 25d were found to be the most active compounds of the series and showed IC50 values of 2.43 µM, 0.96 µM, 1.62 µM, and 3.55 µM, respectively, compared to the standard reference drug Amphotericin B (IC50 = 0.060 µM). All compounds were assessed for Leishmania DNA topoisomerase type IB inhibition activity using the standard drug Camptothecin, and 24a, 24e, 24f, and 25d showed potential results. In order to further validate the experimental results and gain a deeper understanding of the binding manner of such compounds, molecular docking studies were also performed. The stereochemistry of the novel functionalized spirooxindole derivatives was confirmed by single-crystal X-ray crystallography studies.


Introduction
According to the World Health Organization (WHO), in 2021, leishmaniasis emerged as an endemic in 99 countries/territories (out of 200 countries/territories), mainly in 4 eco-epidemiological provinces worldwide (the Americas, East Africa, North Africa, and West and South East Asia) [1,2]. It is caused by Leishmania, a protozoan parasite from the Trypanosomatidae family, which is transmitted by vectors and causes cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL); these are characterized by skin ulcers affecting the mouth, nose, and throat and "kala-azar", respectively [3]. Kala-azar (visceral leishmaniasis) is the fatal form of the disease and is triggered by Leishmania donovani, an intramacrophage protozoan parasite transmitted by the bite of infected female phlebotomine sandflies. This lethal disease affects millions of individuals living in tropical/subtropical regions worldwide [4]. Approximately, twenty-one protozoan parasite species of Leishmania are responsible for causing leishmaniasis, and this is linked to a variety of symptomatology ranging from minor skin lesions at the bite site to the deadly visceral forms. A few standard drugs are available for the treatment of this disease, such as pentavalent antimonials, amphotericin B, its liposomal encapsulation (lamb-liposomal amphotericin B), and miltefosine. Amphotericin B emerged as an alternative second-line treatment for visceral, mucocutaneous, and cutaneous leishmaniasis, especially in the case of human HIV coinfection after resistance was reported in antimonials. In Thailand, amphotericin B is the only effective drug available for the treatment of leishmaniasis [5]. According to published research, there is no current safe and effective treatment for leishmaniasis. The antileishmanial medicines used to treat leishmaniasis at present are accompanied by various kinds of side effects, toxicity, and drug resistance [6,7]. As per the WHO report, approximately 700,000 to 1,000,000 new cases are reported every year [8]. Therefore, there is an urgent need for the advent of effective medications against this neglected tropical disease (NTD).
It has been well-documented that several pharmacologically privileged molecules can be assembled into a single structurally complex molecule with more multi-faceted and enhanced biological activities that can target biological sites of interest in a specific manner to combat specific diseases [40,41]. Several biologically active alkaloidal classes of heterocycles have been reported in the literature and show promising antileishmanial activity in vitro, ex vivo, and in vivo [42][43][44][45]. Recent studies have revealed that several substituted spirooxindoles 15-19 [46][47][48][49] show promising antileishmanial activity against promastigotes and the amastigotes forms of Leishmania (L.) species either in vitro or in vivo when treated with pentamidine, amphotericin B, or miltefosine as one of the standard drugs. Therefore, in our endeavor to search for novel bio-heterocycles as antileishmanial agents, we designed Prototype A, i.e., functionalized spiro[indoline-3,2 -pyrrolidin]-2-one/spiro[indoline-3,3pyrrolizin]-2-one incorporating subunits of 15-19 (Figure 2), and we assessed in vitro antileishmanial activities against the promastigotes form of L. donovani, with the expectation that a new series of amino-acid-based spirooxindole derivatives would also show promising in vitro activity. So far, a literature review has revealed that there is no report available showing amino-acid-based spirooxindoles as antileishmanial agents.  Microwave-assisted organic synthesis (MAOS) is a non-conventional, eco-friendly source of energy in chemical synthesis that can perform the reaction in a shorter time with less energy and furnish the product in a greater yield with higher purities as compared to traditional synthetic processes [50][51][52][53][54][55]. This fascinating method has a wide range of applications in drug discovery evaluation and the pharmaceutical segment for chemical synthesis. It has established an ongoing position in analytical and organic laboratory praxis [56]. Multi-component reactions (MCRs) via 1,3-dipolar cycloaddition reactions have been considered the best potential way for the synthesis of a library of spirooxindole derivatives [57,58].

Synthesis
The 1,3-dipolar cycloaddition reaction of azomethine ylides is a versatile reaction and is well known for the assembly of numerous varieties of complex bioactive azaheterocyclic skeletons [59][60][61]. The azomethine ylide is also reported to serve as an important building block for the construction of several natural-product-inspired aza-heterocycles [62][63][64] and bioactive molecules [65].
We commenced our synthetic investigation by taking isatin 20a, chalcone 21a, and Lproline 22a as starting materials for carrying out microwave-assisted synthesis of spirooxindolepyrrolidine 23a. Initially, the reaction was attempted under refluxing conditions. Therefore, the reaction was carried out by taking 20a (1 equiv.), 21a (1 equiv.), and 22a (1 equiv.) in MeOH under refluxing conditions for 120 min. We were delighted to get the desired spiro compound 23a in 86% yield ( Table 1, entry 1). Then, we analysed the effect of the number of equivalents of the starting materials. Thus, the reaction was repeated in MeOH with 20a (1 equiv.), 21a (1.5 equiv.), and 22a (1.5 equiv.) under refluxing conditions for 180 min, yielding 23a in 96% yield (Table 1, entry 2). It was noticed that changing the number of equivalents led to an improvement in the yield of the reaction. In order to reduce the time to complete the reaction, the reaction was subjected exactly to the same conditions as mentioned in entry no. 2 and allowed to run for 120, 60, and 30 min, which produced 23a in 89%, 67%, and 58% yields, respectively ( It is well known that microwave irradiation has been used as a fundamental tool for constructing aza-heterocycles with interesting properties, either in homogeneous or heterogeneous liquid reaction systems [66]. Utilizing the dual potential of both microwave irradiation as well as the 1,3,-dipolar cycloaddition reaction strategy; equimolar amounts of 20a (1 equiv.), 21a (1.5 equiv.), and 22a (1.5 equiv.) dissolved in MeOH were treated under microwave irradiation conditions at 80 • C for 1 and 3 min, which produced 23a in 41% and 71% yield, respectively (Table 1, entries [10][11]. Intriguingly, when the same reaction was subjected to 5 min under microwave conditions; 23a was obtained in 98% yield (Table 1, entry 12). The reactions were further screened with different solvents (AcCN, ethylene glycol, and ethanol) utilizing the same conditions as mentioned in entry no. 12 with varying times and temperatures (Table 1, entries [13][14][15][16][17][18][19][20][21]. However, none of the reactions produced better yields than those obtained in entry no. 12. Therefore, equimolar amounts of 20a (1 equiv.), 21a (1.5 equiv.), and 22a (1.5 equiv.) dissolved in MeOH under microwave irradiation conditions at 80 • C for 5 min was found to be the best optimized reaction condition (Table 1, entry 12). Substituted isatins 20a-h, substituted chalcones 21a-f, and various amino acids 22a-c were subjected to microwave-assisted 1,3-dipolar cycloaddition reactions in MeOH at 80 • C for 5 min, which produced the desired chalcone-isatin-amino-acid-based spirooxindole compounds 23a-f, 24a-f, and 25a-g in excellent yields (up to 98%) in a highly diastereoselective manner (Scheme 1, Please see Supplementary Materials). In this reaction, [3 + 2] cycloaddition of substituted chalcones occurred with in situ generated azomethine ylides from microwave-assisted decarboxylative condensation of substituted isatins and various secondary amino acids. The physico-chemical data, such as melting point and yield, under conventional conditions as well as in microwave-assisted conditions for all the compounds (23a-f, 24a-f, and 25a-g) are shown in Table 2.
The structures of all the synthesized compounds were well characterized by FT-IR, optical rotation, 1 H-NMR, 13 C-NMR spectroscopy, and HRMS mass spectrometric analysis (Please see Supplementary Materials). Finally, the stereochemistry of the four chiral centres of the cycloaddition reaction was unequivocally determined by single-crystal X-ray diffraction analysis of the cycloadduct 23f ( Figure 3, please see Supplementary Materials). After screening over the series of other derivatives, we found that the 23f prepared in one step and obtained as an off-white solid in 86% yield, which was further subjected to crystallization; we were able to isolate the 23f in~10-11% yield using a slow evaporation crystallization technique with DCM as a solvent at low temperature. After couple of weeks, we came up with the single-crystal X-ray structure of the 23f, the raw data of 23f were subjected to the solution using Olex2 [67], and the crystal was crystallized in a trigonal system in R-3 space group. Consequently, the three-dimensional representation of compound 23f shows that the compound has four chiral carbons, with one carbon having an R-configuration and the other three having S-configurations. The crystal structure confirmed that the trans-geometry of chalcone and the regioselectivity were also well established as a result of the concerted reaction of chalcones with the ylides. The structures of all the synthesized compounds were well characterized by FT-IR, optical rotation, 1 H-NMR, 13 C-NMR spectroscopy, and HRMS mass spectrometric analysis (Please see Supplementary materials). Finally, the stereochemistry of the four chiral centres of the cycloaddition reaction was unequivocally determined by single-crystal X-ray diffraction analysis of the cycloadduct 23f ( Figure 3, please see Supplementary materials). After screening over the series of other derivatives, we found that the 23f prepared in one step and obtained as an off-white solid in 86% yield, which was further subjected to crystallization; we were able to isolate the 23f in ~10-11% yield using a slow evaporation crystallization technique with DCM as a solvent at low temperature. After couple of weeks, we came up with the single-crystal X-ray structure of the 23f, the raw data of 23f were subjected to the solution using Olex2 [67], and the crystal was crystallized in a trigonal system in R-3 space group. Consequently, the three-dimensional representation of compound 23f shows that the compound has four chiral carbons, with one carbon having an R-configuration and the other three having S-configurations. The crystal structure confirmed that the trans-geometry of chalcone and the regioselectivity were also well established as a result of the concerted reaction of chalcones with the ylides.

Single-Crystal X-ray
Furthermore, it was observed that the crystallized framework had a hexagonal architecture consisting of six molecules in a circular fashion around the disordered functionality that takes non-planar circular conformations with the presence of short contacts in the alternate configuration. The molecular arrangement of 23f, its inside functionality in a large cavity, and its size are directly proportional to the distance between the carbon atoms at the opposite sides and the Van der Waals radius of the carbon atom present in the ring. In order to understand more about the intermolecular interactions of 23f, a Hirshfeld surface analysis using Crystal Explorer 3.1 software suite was used [68]. The

Single-Crystal X-ray
Furthermore, it was observed that the crystallized framework had a hexagonal architecture consisting of six molecules in a circular fashion around the disordered functionality that takes non-planar circular conformations with the presence of short contacts in the alternate configuration. The molecular arrangement of 23f, its inside functionality in a large cavity, and its size are directly proportional to the distance between the carbon atoms at the opposite sides and the Van der Waals radius of the carbon atom present in the ring. In order to understand more about the intermolecular interactions of 23f, a Hirshfeld surface analysis using Crystal Explorer 3.1 software suite was used [68]. The 3D representation of short intermolecular contact can be provided by de and di mapped on the Hirshfeld surface, which corresponds to exterior and interior distances, respectively. The d norm , shape index, and curvedness of 23f roughly indicating the presence of strong intermolecular short contacts and stronger Van der Waals interactions. The 2D finger plot of 23f reveals significant interactions corresponding to C-C, H-C, and H-H contributes 4.5%, 8.9%, and 39.0% of the total Hirshfeld surface, respectively, which is again attributable to the presence of strong intermolecular interactions; these are more prominent that of π-π interactions ( Figure 4). As can be seen from Scheme 1 and Figure 4, all synthesized spirooxindoles 23a-f, 24a-f, and 25a-g were obtained in the 71-98% yield range. It was noticed that the reactions were occurring smoothly under microwave conditions with very good to excellent yields; however, the effects of the electron-donating group (EDG) and/or the electronwithdrawing group (EWG) had a marginal influence on the yield of the reaction. Among the spiro[indoline-3,2 -pyrrolidin]-2-one/spiro[indoline-3,3 -pyrrolizin]-2-one derivatives, i.e., 23a-f, 24a-f, and 25a-g, it was noticeable that the EDG (Me, OMe, cycloalkyl, Cl, Br, and I), either on isatin or on Chalcone, produced the desired compound in excellent yield for 23a, 23d, 24d, 24f, and 25a-b. However, in the case of EWG (NO 2 , F), either on isatin or on chalcone, the target compounds were obtained in a good-to-excellent yield range ( Figure 5).

Biological Activity
Considering the importance of amphotericin B in the control of visceral leishmaniasis, the drug was selected as a control in the present study [69]. Camptothecin, a recognized inhibitor of LTopIB, effectively inhibits topoisomerase IB [70].Therefore, both drugs were used as control drugs for performing in vitro antileishmanial activity of all synthesized spirooxindoles 23a-f, 24a-f, and 25a-g.

In Vitro Antileishmanial Activity
The compounds (23a-f, 24a-f, and 25a-g) were initially screened for their in vitro antileishmanial activity against promastigotes of Leishmania donovani (MHOM/IN/1983/AG83) utilizing the Trypan blue dye exclusion method [71] and the plasmid relaxation assay using amphotericin B and camphothesin as standard reference drugs, respectively [72,73].

Trypan Blue Dye Exclusion Method
The promastigotes were harvested from the culture vials, counted, and 2 × 10 6 cells/well were seeded in a 48-well culture plate. The antileishmanial screening of all the derivatives, 23a-f, 24a-f, and 25a-g, as well as the positive control, was performed at various concentrations (2 µg/mL, 4 µg/mL, 8 µg/mL, and 16 µg/mL) added in triplicate. The plate was incubated at 22 ± 1 • C in the BOD incubator for 72 h. After 72 h, each well was counted for the number of viable parasites using the Trypan blue dye exclusion method, and the percentage growth inhibition was calculated by using the formula:

of viable cells in treated well
No. of viable cells in blank well × 100 Percentage growth inhibition = 100 − percentage viability The IC 50 (inhibitory concentration at which 50% of the parasites were dead) value was obtained by plotting a linear dose-response curve in SPSS software (Version 23) [71].

Plasmid Relaxation Assay
The relaxation of supercoiled plasmid DNA is the method used for the determination of LTopIB activity. Various doses of each compound were treated with one unit of pure LTopIB (the enzyme to relax 0.5 µg of supercoiled DNA for 30 min at 37 • C) for 20 min at 4 • C. The reaction mixture, including 0.5 µg of supercoiled pBluescript SK(−) plasmid, 10 mM Tris-HCl buffer pH 7.5, 5 mM MgCl 2 , 0.1 mM EDTA, 15 µg/mL of bovine serum albumin, and 150 mM KCl, was then added in a final volume of 20 µL. After 30 min at 37 • C, the reaction mixtures were stopped by adding 4 µL of loading buffer, which included 5% sarkosyl, 0.12% bromophenol blue, and 25% glycerol. By electrophoresis, the topoisomers were separated on 1% agarose gels and electrophoresed at 2 V/cm for 16 h in a 0.1 M Tris-borate-EDTA buffer (pH 8.0) after being stained with ethidium bromide (0.5 µg/mL). Plotting the percentage of supercoiled DNA versus drug concentrations allowed researchers to determine the 50% inhibition concentration (IC 50 ) values of LTopIB inhibition as the 50% reduction of supercoiled DNA [72].

Inhibition of Leishmanial DNA Topoisomerase IB
Because of the presence of spirooxindole systems in the structure of these compounds, we aimed to assess their inhibitory potential on purified recombinant LTopIB measuring the relaxation of supercoiled plasmid DNA. In this regard, all spirooxindole derivatives were assessed for LTopIB inhibition through the prevention of DNA relaxation in a circular DNA plasmid. All compounds were tested at a single concentration of 100 µM to discard those that did not prevent DNA relaxation by LTopIB. After this initial test, potential inhibitor dose/response curves were performed to obtain their IC 50 values. The compounds 24a, 24e, 24f, and 25d were potent LTopIB inhibitors, and the IC 50 values of all nineteen compounds are shown in Table 3. The lowest IC 50 value corresponded to 24e (IC 50 = 15.7 µM). 23a-f, 24a-f, and 25a- All nineteen compounds exhibited moderate-to-good antileishmanial activity against Leishmania donovani. The results are shown in Table 3.

Structure-Activity Relationship (SAR) Studies
The inhibitory concentration (IC) values for all the spiro[indoline-3,2 -pyrrolidin]-2one/spiro[indoline-3,3 -pyrrolizin]-2-one derivatives, i.e ., 23a-f, 24a-f, and 25a-g, and the positive-control drugs, were also determined against promastigotes of Leishmania donovani utilizing the Trypan blue dye exclusion method [68]. Amphotericin B was taken as a positive control. As can be seen from Table 3, compound 24e, the most active compound of the series, showed potent in vitro antileishmanial activity, with the IC 50 value of 0.96 µM against Leishmania donovani. Compound 24f, the next most active compound in the series (IC 50 = 1.62 µM), exhibited potent antileishmanial activity in comparison to the standard drug Amphotericin B (IC 50 = 0.060 µM). Subsequently, compounds 24a and 25d also showed promising antileishmanial activity, with IC 50 values of 2.43 µM and 3.55 µM, respectively. Furthermore, compounds 23d and 24b showed moderate activity (IC 50 ≤ 10 µM). The rest of the compounds exhibited a lesser activity profile. Thus, SAR experiments indicated that the L-phenylalanine-based spirooxindoles showed a better antileishmanial activity profile as compared to L-proline and L-tryptophan-based counterparts. In proline-based spirooxindoles 23a-f, the EDG group (OMe, Me) on the isatin moiety and the halogen (X = Br) on the chalcone functionality, i.e., 23d, provided significant activity compared to Amphotericin B. Subsequently, phenylalanine-based spirooxindoles 24a-f were found to be the best active compounds among the series. However, the presence of EDG (OMe, Me), EWG (NO 2 , F), or a halogen (X = Br, I) on the isatin moiety showed promising antileishmanial activity in compounds 24a, 24b, 24e, and 24f despite having EDG, EWG, or halogen groups on chalcone, except 24c-d. Furthermore, among tryptophan-based spirooxindoles 25a-g, the presence of EDG (OMe, Me), EWG (NO 2 , F), or a halogen (X = F, Br) on the isatin moiety showed moderate activity (25d) despite having EDG (OMe, Me, cyclohexyl), EWG (X = F), or a halogen (X = F, Cl, Br) on chalcone functionality. It was also observed that the presence of EDG or EWG on the spiroskeleton had no influence on the yield of the reaction.

Protein Preparation
The protein crystal 3D structures of the heterodimeric L. Donovani topoisomerase I-vanadate DNA complex were taken from the protein data bank (PDB), PDB ID 2B9S. The protein was minimized using the simulation protocol via the CHARMm-based smart minimizer method, and protein preparation involved five different steps: cleaning protein, inserting missing atoms, refining loops, minimizing loops, and protonating protein.

In Silico Studies
Analysis of the docking results was carried out by comparing the binding affinities of all the proposed docked molecules to the complex protein. The docking of the abovementioned protein was carried out by removing DNA, and the remaining protein was kept in a grid box. Then, we explored the binding orientation of active functionalized spirooxindoles in terms of their Cdocker energy and Libdock score. It is to be noted that low Cdocker energy and high Libdock score values indicate higher binding affinity toward the target protein, thereby reflecting its higher potency (Table 4). The docking results for 24a against Leishmania donovani showed a high binding affinity docking score indicated by a total score of 128.598, and it formed three H-bonds of length 2.19 Å, 2.9 Å, and 2.16 Å to the hydrophobic nucleophilic residues, i.e., the side chains of ASP: A-353 (aspartic acid), ARG: A-190 (arginine), and ASN: B-221 (asparagine), respectively. In the docking pose of the complex, the chemical nature of binding site residues within a radius of 3 Å showed non-bonding Van der Waals interactions with HIS: A-193 (histidine), ARG: A-314 (arginine), THR: B-217 (threonine), ILE: B-220 (isoleucine), and LYS: A-352 (lysine), thus leading to more stability and activity in this compound. In addition, 24a also exhibited a π-anion interaction with ASP: A-353 and an alkyl-π-alkyl interaction with the TYR: B-222 (thyrosine) amino acid residue ( Figure 6).  The π-anion interaction was observed between LYS A:352 (lysine) and isatin of bond length 3.85 Å. The conventional H-bonding of amino acids ASP: A-353 (aspartic acid) and ARG: A-190 (arginine) with bond lengths of 3.52 Å and 2.43 Å were present along with NH and carbonyl group moieties of ligand 24f, respectively. Subsequently, 24f also showed other interactions involving carbon-hydrogen bonds as well as π-cation, π-anion, and π-alkyl interactions ( Figure 8   The docking results for camptothecin against Leishmania donovani showed a high binding affinity docking score indicated by a total score of 123.320 and formed two conventional H-bonds of length 2.55 Å and 1.79 Å to the hydrophobic nucleophilic residues, i.e., the side chains of ALA: A-324 (alanine) and LYS: A-319 (lysine), respectively. In the docking pose of the complex, the chemical nature of binding site residues showed non-bonding Van der Waals interactions with PHE: A-187 (phenylalanine), GLY: A-189 (glycine), THR: A-326 (threonine), SER: A-354 (serine), and GLU: A-353 (glutamic acid), which give extra stability and activity in this compound. In addition, camptothecin also exhibited the π-cation interaction with ARG: A-190 (arginine) and the alkyl-π-alkyl interaction with ALA: A-324 (alanine) and HIS: A-193 (histidine) amino acid residues ( Figure 10).